1. Field of the Invention
The present invention relates to a method for identifying a polymorphism more sensitively and more accurately than prior art methods.
2. Description of Related Art
With the recent progress in genetic engineering technologies and gene recombination technologies, genetic tests through nucleic acid analyses have been widely used in applications to medical services, researches, and industries. Such tests are to detect the presence of DNA which has a target nucleotide sequence within a sample, and have been applied not only to diagnosis and treatment of diseases, but also to food inspection and other various fields. In particular, a genetic polymorphism such as a SNP (Single Nucleotide Polymorphism) is considered to be a major factor contributing to the individual difference in the vulnerability against a specific disease such as cancer, the drug metabolizing capacity, and so forth. Genetic polymorphism analyses have been widely conducted not only in academic researches but also in actual clinical tests. Therefore, highly accurate and quick methods for detecting a genetic polymorphism have been enthusiastically developed.
As to the method for detecting and identifying a genetic polymorphism, there are many reported methods in which artificially synthesized polynucleotides such as probes and primers are used to examine the nucleotide sequences of nucleic acids. For example, some methods are to analyze the nucleotide sequence of a SNP serving as the analysis target and its neighboring region by molecular-biological enzymatic reactions. Such methods can be exemplified by: a method in which a region including a polymorphism such as a SNP can be detected by PCR (Polymerase Chain Reaction) amplification; and a method in which a SNP can be detected by a ligation reaction using a probe including the detection target SNP at the 3′ end and a probe including a nucleotide adjacent to the 5′ side of the SNP, at the 5′ end, and subsequent determination regarding the obtainability of a polynucleotide bound with these two probes.
In particular, often employed SNP analysis methods are SSP-PCR (Sequence Specific Primer-PCR) method and ASP-PCR (Allele Specific Primer-PCR) method, in which a SNP can be detected by PCR using a primer specifically bindable to a specific nucleotide sequence, allele, and the like, and subsequent determination regarding the presence/absence of the PCR product. The reason is that, since the detection and recognition of a nucleotide sequence (genetic polymorphism) can be carried out concurrently with enhancement of its signal, the polymorphism detection by means of the SSP/ASP-PCR method can enable the SNP detection even in the case where only a small amount of specimen is available, or the case where the nucleic acid concentration in a sample is very low, like a case of a specimen in a clinical test, and therefore these methods are very useful.
There are many reported SNP analysis methods using no amplification reaction, unlike the SSP-PCR method and the ASP-PCR method. For example, there is disclosed a method (1) for detecting a SNP by using (a) a partially-double-stranded nucleic acid molecule comprising: a single-stranded nucleic acid molecule complementary to a nucleic acid molecule as a detection target; and (b) one or two single-stranded nucleic acid molecule(s) hybridizing with a part of the single-stranded nucleic acid molecule (a), wherein regions of single-stranded structure of the partially-double-stranded nucleic acid molecule are complementary to regions including the identification site of the nucleic acid molecule as the detection target (for example, refer to Patent Document 1). Under the coexistence of the partially-double-stranded nucleic acid molecule and the nucleic acid molecule as the detection target, the single-strand of the nucleic acid molecule of the invention and the target nucleic acid molecule are replaced by the strand displacement reaction, thereby forming a double-stranded structure of the long chain of the nucleic acid molecule of the invention and the target nucleic acid molecule. The target nucleic acid molecule can be detected by detecting this double-stranded structure.